Laboratory investigations in Hereditary Spherocytosis

LABORATORY INVESTIGATIONS IN HEREDITARY SPHEROCYTOSIS (HS)
  • Peripheral blood picture –
    • Abnormal small, dark staining red blood cells which lack central pallor – spherocytes
    • Reticulocyte count is increased
  • Other hematological findings
    • Hemoglobin – decreased
    • Mean corpuscular hemoglobin concentration – increased
    • Red cell distribution width – increased
  • Additional tests are
    • Osmotic fragility test
      • increased osmotic fragility and incubated osmotic fragility tests
      • RBC fragility is increased when RBC’s have decreased surface area to volume ratios
      •  Procedure: Standard volume of fresh,heparinized blood is mixed with NaCl solution ranging from 0.85% (isotonic saline) to 0.0% (distilled water) in 0.05 to 0.1% increments. After 30 minutes incubation at room temperature, the tubes are centrifuged & the absorbance of the supernatant is measured spectrophotometrically at 540nm.
      • Incubating the blood at 370 C for 24 hrs before performing the test (called incubated osmotic fragility test) allows hereditary spherocytosis cells to become more spherical which is needed to detect mild case
      • Major draw back of this test is lack of sensitivity and specificity. It cannot differentiate between Hereditary spherocytosis and spherocytes in other conditions such as immune hemolytic anemias, burns and other acquired causes.
    • Eosin- 5′-Maleimide (EMA) binding test
      • EMA binding test is more sensitive alternative for confirmation of HS
      • EMA is fluorescent dye that binds to transmembrane protein band 3, Rh, Rh Ag & CD47 in RBC membrane. 
      • When measured in a flow cytometer, specimen from HS have lower mean fluorescene intensity (MFI)  than the normal RBC’s. Result is expressed as % of decrease in MFI. When the patient specimen is compared to  normal controls.
    • SDS-PAGE analysis
      • Sodium Dodecyl Sulfate – Polyacrylamide gel electrophoresis (SDS-PAGE) can be used to identify the protein deficiency by separating various proteins using Gel electrophoresis
  • Direct antiglobulin test – negative
  • Parametres indicating hemolysis
    • Serum haptoglobulin – decreased
    • Serum lactate dehydrogenase – increased
    • Serum indirect bilirubin – increased
                               
Reference:
  • Elaine M.Keohane.Intrinsic defects leading to increased erythrocyte destruction.In :Elaine M.Keohane, Larry J Smith, Jeanine M.Walenga. Rodaks hematology. Clinical principles & applications chapter 24;367-393.